DGK α and ζ Activities Control TH1 and TH17 Cell Differentiation

CD4+ T helper (TH) cells are critical for protective adaptive immunity against pathogens, and they also contribute to the pathogenesis of autoimmune diseases. How TH differentiation is regulated by the TCR's downstream signaling is still poorly understood. We describe here that diacylglycerol kinases (DGKs), which are enzymes that convert diacylglycerol (DAG) to phosphatidic acid, exert differential effects on TH cell differentiation in a DGK dosage-dependent manner. A deficiency of either DGKα or ζ selectively impaired TH1 differentiation without obviously affecting TH2 and TH17 differentiation. However, simultaneous ablation of both DGKα and ζ promoted TH1 and TH17 differentiation in vitro and in vivo, leading to exacerbated airway inflammation. Furthermore, we demonstrate that dysregulation of TH17 differentiation of DGKα and ζ double-deficient CD4+ T cells was, at least in part, caused by increased mTOR complex 1/S6K1 signaling.


INTRODUCTION
CD4 + T helper (T H ) cells play a central role in orchestrating adaptive immune response to pathogens and also contribute to autoimmune diseases (1,2). After antigen stimulation, naïve CD4 + T cells differentiate into discrete subsets of effector T H cells with distinct functions and cytokine profiles. Interferon-γ (IFN-γ)-producing T H 1 cells, induced by IL-12 and directed by transcriptional factor T-bet, are critical for the clearance of intracellular pathogens (3,4). T H 2 cells, which secrete IL-4, IL-5, and IL-13 and are controlled by GATA-3, are crucial for protection against parasites and extracellular pathogens (5,6). T H 17 cells produce IL-17A, IL-17F, and IL-22, and play an important role in the control of specific pathogens such as fungi. T H 17 differentiation is driven by a combination of TGF-β and IL-6 and requires transcriptional factor RORγt and RORα. IL-23 promotes T H 17 responses by enhancing their survival and stabilization (7)(8)(9)(10)(11)(12).
Despite their importance in host immunity against pathogens, T H cells can be pathogenic and contribute to various diseases. Both exaggerated and defective T H 1 response has been linked to the induction of autoimmune diseases (13)(14)(15). T H 2 cells contribute to allergies and asthma (16,17). T H 17 cells are associated with many autoimmune and inflammatory diseases such as psoriasis, inflammatory bowel diseases, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis (8,11,(18)(19)(20). Thus, understanding how T H responses are regulated is important to manipulate immune responses, to improve host defense against microbial infection, and to treat autoimmune diseases.
Engagement of the TCR on naïve CD4 + T cells is essential for their activation and further differentiation to T H cells (21,22). Evidence has revealed that TCR signal strength and downstream signaling pathways as well as cytokine and costimulatory signals shape T H lineage differentiation (23)(24)(25)(26). A critical event after TCR engagement is the generation of the second messenger diacylglycerol (DAG) by activated PLCγ1. DAG associates with and allosterically activates RasGRP1 and PKCθ, leading to the activation of the Ras-Erk1/2-AP1 and PKCθ-IKK-NFκB signaling pathways, respectively, and is indispensable for T cell activation (27)(28)(29)(30). Since it has been demonstrated that both Ras-and PKCθ-mediated signal cascades are involved in T H differentiation (31)(32)(33)(34), it is important to investigate if DAG concentrations should be tightly controlled during T H differentiation.

Mice
DGKα −/− , DGKζ −/− , and ERCre mice were generated as previously described (38,39,56). DGKζ f /f mice were generated by introducing two LoxP sites that flank exons 10-14 of the Dgkz locus (57). TCR transgenic OT2 mice were purchased from the Jackson Laboratory and were cross-bred with DGKα −/− ζ f/f ERCre mice to generate DGKα −/− ζ f/f OT2 ERCre mice in specific pathogen-free facilities at Duke University Medical Center. The experiments in this study were performed according to a protocol approved by the Institutional Animal Care and Usage Committee of Duke University. DGKα −/− ζ f/f or DGKα −/− ζ f/f OT2 ERCre mice were intraperitoneally injected with tamoxifen (100 mg/kg body weight) on the first, second, and fifth day to delete DGKζ, and mice were then euthanized for experiments on the eighth day.

Flow Cytometry
Standard protocols were used to prepare single cell suspensions from the spleen and lymph nodes of mice (in IMDM containing 10% FBS and antibiotics). Red blood cells were lysed using an ACK buffer. Samples were subsequently stained with antibodies in PBS containing 2% FBS and collected on a BD FACSCanto II cytometer. Intracellular staining for T-bet and RORγt was performed using the eBioscience Foxp3 Staining Buffer Set. Intracellular staining for IFNγ, IL-4, IL-17A, and IL-17F was performed using the BD Biosciences Cytofix/Cytoperm and Perm/Wash solutions.
Adoptive Transfer, Immunization, and Airway Inflammation

Real-Time RT-PCR
Cells were lysed in Trizol for RNA preparation. The first strand cDNA was made using the iScript Select cDNA Synthesis Kit (Biorad). Real-time quantitative PCR was conducted using Eppendorf realplex 2 . Expressed levels of target mRNAs were normalized with β-actin and calculated using the 2 − CT method. Primers used in this study are listed as following:

Statistical Analysis
Data are presented as mean ± SEM, and statistical significance was determined by two-tailed Student's t-test. The p-values are defined as follows: * p < 0.05, * * p < 0.01, * * * p < 0.001.

Deficiency of Either DGKα or ζ Impaired T H 1 Cell Differentiation
DGKα and ζ are dynamically regulated during T cell development and activation (27,35,39,40). We found that DGKα mRNA was decreased in T H 0, T H 1, T H 2, T H 17, and iTregs compared with naïve CD4 + T cells. DGKζ mRNA also was decreased in T H 0, T H 1, and T H 17 cells but not in T H 2 and iTregs compared with naïve CD4 + T cells ( Figure 1A). Both DGKα and ζ appeared more significantly down-regulated in T H 1 and T H 17 conditions than in T H 0 condition. To examine the role of DGKα and ζ in T H differentiation, WT, DGKα −/− , and DGKζ −/− CD44 − CD62L + naïve CD4 + T cells were cultured in T H 1, T H 2, and T H 17 polarization conditions in vitro for 4-5 days. DGKα −/− or DGKζ −/− CD4 + T cells displayed impaired differentiation to T H 1 cells, which was indicated by decreases of IFN-γ + cells in both percentages and numbers (Figures 1B,C), IFN-γ concentration in culture supernatants (Figure 1F), and IFN-γ mRNA levels ( Figure 1G), accompanying the decreased expression of T-bet ( Figure 1H). However, total CD4 + T cells numbers were increased in the absence of either DGKα or ζ during T H 1 polarization (Figure 1C), suggesting that impaired T H 1 differentiation of DGKα −/− or DGKζ −/− CD4 + T cells did not result from decreased expansion. In contrast, T H 2 and T H 17 differentiation was not obviously affected by DGKα or ζ deficiency. This was reflected by similar percentages of IL-4 + or IL-17 + cells (Figures 1B,D,E) and similar levels of IL-4 or IL-17A proteins in culture supernatants ( Figure 1F) and mRNAs (Figure 1G), which correlated with comparable expression of GATA-3 or RORγt (Figure 1H). Both DGKα −/− CD4 + T cells and DGKζ −/− CD4 + T cells displayed slightly improved survival under the T H 1 condition and had similar survival rates under T H 2 and T H 17 conditions (Figure 1I), suggesting that their reduced T H 1 responses were not due increased cell death. Together, these data suggested individual DGKα and DGKζ are required for T H 1 differentiation, but are dispensable for T H 2 and T H 17 development in vitro.

Deficiency of Both DGKα and ζ Promoted T H 1 and T H 17 Differentiation
DGKα and ζ promote T cell and iNKT cell maturation synergistically in the thymus (52,54). To determine if DGKα and ζ exert a synergistic role during T H differentiation, we generated DGKα −/− ζ f/f -ERCre (DKO) mice so that both DGKα and ζ were ablated after tamoxifen-induced deletion of DGKζ. In contrast to DGKα or ζ single-knockout T cells, DKO CD4 + naïve T cells showed enhanced capacity to differentiate into both T H 1 and T H 17 cells but similar T H 2 differentiation compared with their WT counterparts (Figures 2A,B), coinciding with increased IFNγ and IL-17A but not IL-4 concentration in culture supernatants ( Figure 2C) and IFN-γ and IL-17A mRNA levels in these cells ( Figure 2D).   (Figures 3A-C). In addition, higher percentages of DKO OT2 T cells expressed IFN-γ, IL-17A, and IL-17F than WT controls following in vitro PMA and ionomycin stimulation for 4 h (Figures 3D-F). Because of increased DKO OT2 T cell numbers, DKO OT2 T H 1 and T H 17 cell numbers were much greater than WT controls in dLNs and particularly in the spleen (Figures 3G,H). Moreover, DKO OT2 T cells contained more IFN-γ-, IL-17A-, and IL-17F-positive cells, which was detected by intracellular staining (Figures 3I,J), and secreted more cytokines to culture supernatants, which was detected by ELISA (Figures 3K,L) (Figures 4A-C). DKO donor-derived OT2 cells in both dLNs and spleens produced more IL-17A and IL-17F as well as IFN-γ in response to in vitro stimulation with PMA and ionomycin for 4 h (Figures 4D-H) or with OVA 323−339 peptide for 2 days (Figures 4I-M). Concordantly, both IFN-γ and IL-17A levels in bronchoalveolar lavage fluid (BALF) were elevated in recipients with DKO OT2 T cells compared with those with WT OT2 T cells ( Figure 5A). Moreover, DKO OT2 cell recipients contained more neutrophils and lymphocytes than those with WT control in BALF (Figures 5B,C) and in interstitial lung tissues that surround the bronchioles ( Figure 5D). Together, these results demonstrated that DGKα and ζ deficiencies in CD4 + T cells exacerbated airway inflammation, likely as a result of enhanced T H 17 responses to protein allergens.

Effects of DGKαζ Double Deficiency on Expression of Critical Lineage Transcription Factors
T-bet, GATA-3, RORγt, and RORα are transcription factors that play critical roles in T H 1, T H 2, and T H 17 differentiation, respectively. Under the T H 1 polarization condition, DKO CD4 + T cells expressed higher levels of T-bet at both mRNA and protein levels than WT controls (Figures 6A,B), which was consistent with their elevated T H 1 responses. In contrast, GATA-3 expression in DKO CD4 + T cells was not obviously different from WT controls under the T H 2 polarization condition (Figure 6C), consistent with a minimal effect of DKO on T H 2 responses as shown in Figure 2. Interestingly, Rorc (gene encoding RORγt) mRNA levels were obviously decreased in DKO CD4 + T cells under the T H 17 polarization condition (Figure 6D), although RORγt protein was only slightly decreased ( Figure 6E). In contrast, RORa mRNA levels were increased in DKO CD4 + T cells 24 and 36 h after polarization ( Figure 6F). Both RORα and RORγt are important for T H 17

Effects of DGKα and ζ Double Deficiency on mTORC1/S6K1 Signaling During T H 1 and T H 17 Cell Differentiation
DGKα and ζ negatively control DAG-mediated Ras-Erk1/2 activation in thymocytes and naïve T cells following TCR engagement (36,38,54). We further examined how DGKα and ζ double deficiency might affect this pathway during T H polarization. As shown in Figure 7A, Erk1/2 phosphorylation was obviously enhanced in DKO CD4 + T cells under T H 0, T H 1, T H 2, and T H 17 conditions, suggesting that DGKα and ζ negatively controlled Erk1/2 activation during effector CD4 + T cell differentiation. Previous studies have found that DAGmediated RasGRP1-Ras-Erk, PI3K-Akt, and PKCθ-CARMA1 pathways participate in TCR-induced mTORC1 activation and DGKα and ζ double deficiency but not DGKα or ζ single deficiency leads to enhanced mTOR signaling in developing thymocytes (36,64,65) and that mTOR plays important roles in Th differentiation (65)(66)(67)(68)(69). Although, S6 phosphorylation, an mTORC1/S6K1-dependent event, in T H 1 cells appeared unaffected by DGKα and ζ double deficiency, it was obviously increased in DKO CD4 + T cells under T H 0, T H 2, and T H 17 polarization conditions, suggesting that DGKα and ζ negatively controlled mTORC1 signaling in T H 0, T H 2, and T H 17 cells. Treatment of WT and DKO CD4 + T cells with either rapamycin or the S6K1 inhibitor PF-4708671 caused about 50% reduction of IFNγ + cells in both cell types but DKO CD4 + T cells still contained higher percentages of IFNγ + cells than WT controls. Thus, DKO CD4 + T cells were partially sensitive to mTORC1/S6K1 inhibition (Figures 7B,C), suggesting that additional mechanisms might contribute to enhanced T H 1 differentiation in these cells. In contrast, T H 17 differentiation of both DKO and WT CD4 + T cells was potently inhibited by either rapamycin or PF-4708671 (Figures 7D,E). Although, we could not rule out potential off-target effects of PF-4708671 and rapamycin, our data suggested that enhanced mTORC1/S6K1 signaling might contribute to the elevated T H 17 responses of DKO CD4 + T cells.
Dysregulated T H 1 and T H 17 responses contribute to the pathogenesis of numerous autoimmune diseases, including psoriasis, inflammatory bowel disease, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, experimental autoimmune encephalomyelitis, and neutrophil-related airway inflammation (8,11,(13)(14)(15)(18)(19)(20). We have shown that dysregulated T H 1 and T H 17 responses in the absence of DGKα and ζ are pathogenic, indicated by exacerbated neutrophil-related airway inflammation. Interestingly, DGKα and ζ double deficiency leads to a loss of T cell tolerance and the development of autoimmune diseases in mice (manuscript in preparation). Enhanced CD4 + T cell effector function might be an important contributor to the development of autoimmune diseases in these mice. Thus, modulating DGKα and ζ activity could be a potential strategy to shape immune responses. Of note, although DGKα and ζ double deficiency does not obviously affect iTreg induction in vitro, our data do not rule out a potential role of DGK activity in peripheral Treg induction from naïve CD4 + T cells in vivo. Additional studies are needed to determine whether DGKα and ζ play a redundant role in Treg cells.
In summary, DGK activity plays selective roles in T H cell differentiation. A single knockout of DGKα or ζ impaired T H 1 cell differentiation whereas a deficiency of both DGKα and ζ promoted T H 1 and T H 17 cell differentiation in vitro and in vivo. Such dysregulated expansion of both T H cells in the absence of DGKα and ζ caused severe airway inflammation. DGKα and ζ double deficiency led to enhanced mTORC1-S6K1 activation during T H 17 cell differentiation, which may contribute to enhanced T H 17 cell differentiation. Our study demonstrated the role of DGKs in T H cell differentiation and provides useful evidence for these enzymes as potential targets for therapeutic approaches of autoimmune diseases associated with the dysregulation of T H 1 and T H 17 cells.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/supplementary material.

ETHICS STATEMENT
The experiments in this study were performed according to a protocol approved by the Institutional Animal Care and Usage Committee of Duke University.

AUTHOR CONTRIBUTIONS
JY designed and performed experiments, analyzed data, and wrote the paper. H-XW, JX, LL, and JW performed experiments and analyzed data. EW generated critical reagents. X-PZ conceived the project, designed experiments, participated in data analysis, and wrote the paper.

FUNDING
This study was supported by the National Institutes of Health (R01AI079088, R01AI101206, and R56AG060984).